A Monte Carlo approach for improving transient dopamine release detection sensitivity

Current methods using a single PET scan to detect voxel-level transient dopamine release—using F-test (significance) and cluster size thresholding—have limited detection sensitivity for clusters of release small in size and/or having low release levels. Specifically, simulations show that voxels with release near the peripheries of such clusters are often rejected—becoming false negatives and ultimately distorting the F-distribution of rejected voxels. We suggest a Monte Carlo method that incorporates these two observations into a cost function, allowing erroneously rejected voxels to be accepted under specified criteria. In simulations, the proposed method improves detection sensitivity by up to 50% while preserving the cluster size threshold, or up to 180% when optimizing for sensitivity. A further parametric-based voxelwise thresholding is then suggested to better estimate the release dynamics in detected clusters. We apply the Monte Carlo method to a pilot scan from a human gambling study, where additional parametrically unique clusters are detected as compared to the current best methods—results consistent with our simulations.     

Hyperphosphatemia,Phosphoprotein Phosphatases,and Microparticle Release in Vascular Endothelial Cells

Hyperphosphatemia in patients with advanced CKD is thought to be an important contributor to cardiovascular risk, in part because of endothelial cell (EC) dysfunction induced by inorganic phosphate (Pi). Such patients also have an elevated circulating concentration of procoagulant endothelial microparticles (MPs), leading to a prothrombotic state, which may contribute to acute occlusive events. We hypothesized that hyperphosphatemia leads to MP formation from ECs through an elevation of intracellular Pi concentration, which directly inhibits phosphoprotein phosphatases, triggering a global increase in phosphorylation and cytoskeletal changes. In cultured human ECs (EAhy926), incubation with elevated extracellular Pi (2.5 mM) led to a rise in intracellular Pi concentration within 90 minutes. This was mediated by PiT1/slc20a1 Pi transporters and led to global accumulation of tyrosine- and serine/threonine-phosphorylated proteins, a marked increase in cellular Tropomyosin-3, plasma membrane blebbing, and release of 0.1- to 1-μm-diameter MPs. The effect of Pi was independent of oxidative stress or apoptosis. Similarly, global inhibition of phosphoprotein phosphatases with orthovanadate or fluoride yielded a global protein phosphorylation response and rapid release of MPs. The Pi-induced MPs expressed VE-cadherin and superficial phosphatidylserine, and in a thrombin generation assay, they displayed significantly more procoagulant activity than particles derived from cells incubated in medium with a physiologic level of Pi (1 mM). These data show a mechanism of Pi-induced cellular stress and signaling, which may be widely applicable in mammalian cells, and in ECs, it provides a novel pathologic link between hyperphosphatemia, generation of MPs, and thrombotic risk.     

Applying attachment theory to effective practice with hard-to-reach youth: the AMBIT approach

Adolescent Mentalization-Based Integrative Treatment (AMBIT) is a developing approach to working with “hard-to-reach” youth burdened with multiple co-occurring morbidities. This article reviews the core features of AMBIT, exploring applications of attachment theory to understand what makes young people “hard to reach,” and provide routes toward increased security in their attachment to a worker. Using the theory of the pedagogical stance and epistemic (“pertaining to knowledge”) trust, we show how it is the therapeutic worker’s accurate mentalizing of the adolescent that creates conditions for new learning, including the establishment of alternative (more secure) internal working models of helping relationships. This justifies an individual keyworker model focused on maintaining a mentalizing stance toward the adolescent, but simultaneously emphasizing the critical need for such keyworkers to remain well connected to their wider team, avoiding activation of their own attachment behaviors. We consider the role of AMBIT in developing a shared team culture (shared experiences, shared language, shared meanings), toward creating systemic contexts supportive of such relationships. We describe how team training may enhance the team’s ability to serve as a secure base for keyworkers, and describe an innovative approach to treatment manualization, using a wiki format as one way of supporting this process.     

Enhanced oxygen consumption in adrenal medulla on stimulation with acetylcholine

When perfused cortex-free ox adrenal medulla was stimulated to secrete catecholamine by infusion of 0.1 mM acetylcholine for 4 min, the oxygen consumption increased to a value which was 0.15 +/- 0.07 mumole O2/min/g wet weight (+/- S.D., N = 12) above the pre-stimulation value of 0.49 +/- 0.15 (P less than 0.001). 1.4 +/- 0.9 (+/- S.D., N = 12) mole of catecholamine was secreted per mole of enhanced O2 consumption in the 16 min following the start of the stimulation. The rate of ATP hydrolysis by the proton-translocating Mg-ATPase of the chromaffin granule may increase on fusing with the plasma membrane of the chromaffin cell during exocytosis. However, from the amount of catecholamine secreted, this was estimated to account for less than 17% of the oxygen consumption increase. The amount of catecholamine secreted by 4 min 0.1 mM acetylcholine stimulations correlated with the enhancement of oxygen consumption (r = 0.82, P less than 0.001) but, on stimulation with 60 microM veratridine for 4 min, O2 consumption enhancement was anomalously low. This dependence on mode of stimulation suggests that ATP consumption in exocytosis itself is an inadequate explanation. Ouabain-sensitive oxygen consumption rose from undetectable levels to 18 +/- 8% (+/- S.D., N = 4) of the basal respiration during prolonged 0.1 mM acetylcholine stimulation in the absence of Ca, indicating that Na,K-ATPase was not responsible for all of the oxygen consumption enhancement.     

Psychiatric problems in the first year after mastectomy

The psychiatric morbidity associated with mastectomy was assessed in 75 women by following them up from the time they presented with suspected breast cancer to one year after the operation. Fifty women with benign breast disease served as controls. Throughout the follow-up period the incidence of psychiatric problems was higher among the women who had undergone mastectomy. One year after surgery 19 (25%) of these women compared with only 5 (10%) of the controls needed treatment for anxiety or depression or both, and 16 (33%) compared with 3 (8%) respectively had moderate or severe sexual difficulties. Altogether 29 patients in the mastectomy group (39%) and six of the controls (12%) had serious anxiety, depression, or sexual difficulties. Of the eight women in the mastectomy group who sought help for their problems, only two felt that the help given had been appropriate. The inability to recognise and treat these emotional disturbances is a common and serious problem. Monitoring by specially trained nurses and social workers might help to identify them earlier and even reduce them.     

Lymphatic mapping with 99mTc-Evans Blue dye in sheep

Objective   ^99mTc-Evans Blue (EB) is an agent that contains both radioactive and color signals in a single dose. Earlier studies in animal models have suggested that this agent when compared with the dual-injection technique of radiocolloid/blue dye can successfully discriminate the sentinel lymph node. The aim of this study was to investigate the potential of ^99mTc-EB as an agent to map the lymphatic system in an ovine model. Methods  Doses of ^99mTc-EB (23 MBq) containing EB dye (4 mg) were administered intradermally to the limbs of four anesthetized sheep, and they were then imaged over 20–30 min using a gamma camera. The study protocol was repeated using ^99mTc-antimony trisulfide colloid (ATC) and Patent Blue V dye. The lymph nodes (popliteal, inguinal, and iliac for hind limbs or prescapular for fore limbs) were identified with a gamma probe during the operative exposure, then dissected and counted in a large volume counter. Results  Simple and complex (dual) drainage patterns were visible on the scans, and the sentinel node was more radioactive than higher tier nodes in a chain, for both radiotracers. For ^99mTc-EB, maximum radioactive uptake was achieved at 3–6 min for popliteal lymph nodes, 12–14 min for iliac nodes, and 13–14 min for prescapular nodes. ^99mTc-ATC resulted in maximum radioactive uptake at 4–6 min for popliteal lymph nodes, 13 min for an inguinal node, 13–20 min for iliac nodes, and 18 min for a prescapular node. Following ^99mTc-EB injection, 15/15 lymph nodes harvested were all radioactive and blue. For ^99mTc-radiocolloid/Patent Blue V injection, 8/14 nodes were radioactive and blue, and 6/14 nodes were radioactive only. Conclusions  The soluble radiotracer ^99mTc-EB appeared to be a useful lymphoscintigraphic agent in sheep, in which radioactive counts from superficial lymphatic channels and lymph nodes were sufficient for planar imaging. In comparison with ^99mTc-antimony trisulfide colloid, both tracers discriminated the sentinel lymph node up to 50 min after administration; however, ^99mTc-EB had the advantage of providing radioactive (gamma probe) and color signals simultaneously during the operative exposure.     

Leucine suppresses acid-induced protein wasting in L6 rat muscle cells

BACKGROUND: Metabolic acidosis induces protein wasting in skeletal muscle cells, accompanied by decreased glycolysis and compensatory increased consumption of other metabolic fuels, implying that protein wasting arises from fuel starvation and might be rectified by fuel supplements. Design To test this hypothesis, total protein and protein degradation (release of 14C-phenylalanine) were measured in L6 skeletal muscle cells cultured in Eagle's Minimum Essential Medium at pH 7.1-7.5 for 3 days with metabolic inhibitors or metabolic fuel supplements. RESULTS: Inducing metabolic fuel starvation with inhibitors (1 mmol L(-1) 2-deoxyglucose or 0.1 mmol L(-1) KCN [potassium cyanide]) failed to stimulate protein degradation or net protein wasting under nonacidaemic conditions (pH 7.5). Conversely metabolic fuel supplements (1 mmol L(-1) octanoate, pyruvate or alanine) failed to increase the protein content of the cultures at any pH tested, in spite of significant consumption of the fuels by the cells. Only leucine (1-3 mmol L(-1)) increased protein content and suppressed protein degradation in opposition to the catabolic effect of acidaemia (pH 7.1). Conclusion Leucine exerts a beneficial anabolic effect on cultured skeletal muscle cells in the face of metabolic acidaemia. The failure of other metabolic fuels to do this, and of the metabolic inhibitors to exert a catabolic effect, suggests that leucine acts as a specific modulator of protein turnover and not as a nonspecific source of carbon for oxidation as a fuel.     

Addition of the 1-cyano-1-methylethyl radical to α-methylstyrene

1-Cyano-1-methylethyl end-groups in copolymers of α-methylstyrene (MST) with methyl methacrylate (MMA) and with styrene (STY) have been examined by ¹³C NMR; the end-groups have been derived from initiators suitably enriched with carbon-13. It has been shown that at 60°C MST closely resembles STY in reactivity towards the 1-cyano-1-methylethyl radical. MST is 1,89 times as reactive as MMA towards the radical at 40°C; the corresponding factors at 60 and 100°C are 1,69 and 1,55, respectively. There is no evidence for reversibility in the reaction of MST with the 1-cyano-1-methylethyl radical.     

Phosphate metabolism in erythrocytes of critically ill patients

Orthophosphate (Pi), adenosine 5'-diphosphate (ADP), adenosine 5'-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) were measured in the erythrocytes of patients in an intensive care unit. The patients' plasma concentration of Pi varied from 0.1 to 4.2 mmol/l, and the corresponding concentration in erythrocytes varied from 0.1 to 2.0 mmol/litre of cells. Marked ATP depletion (less than 1 mmol/litre of cells) was only observed when erythrocyte Pi was less than 0.3 mmol/litre of cells and plasma Pi was less than 0.35 mmol/l. No dependence of 2,3-DPG concentration on the cellular concentration of Pi was detected. The phosphorylation potential [ATP]/([ADP] X [Pi]) varied inversely with the erythrocyte concentration of Pi. Hence the calculated free energy of hydrolysis of ATP in the cell increased from -58 kJ/mol in the most hypophosphataemic samples to -51 kJ/mol in the most hyperphosphataemic. Such changes may adversely affect cell function by altering the steady state mass-action ratios of ATPase reactions. When erythrocytes from normal donors were incubated in solutions containing 1 or 5 mmol/l Pi, the cellular concentrations of Pi stabilized at 1.09 and 2.85 mmol/litre of cells respectively. The corresponding rates of lactate production were 2.09 and 3.11 mmol h-1 litre-1 of cells. In spite of this stimulation of glycolysis (and hence of the flux through ATP synthesizing steps of the Embden-Meyerhof pathway), no significant change in ATP concentration was observed. As in the patients' cells, this indicates that, when extracellular Pi concentrations are perturbed, the concentrations, in erythrocytes, of organic phosphates are more closely regulated than the concentration of Pi.     

A study of intracellular orthophosphate concentration in human muscle and erythrocytes by 31P nuclear magnetic resonance spectroscopy and selective chemical assay

In order to study the relationship between extracellular and intracellular concentrations of orthophosphate (Pi), phosphorus nuclear magnetic resonance spectra were recorded, at rest, from the flexor digitorum superficialis muscle of hypophosphataemic patients with vitamin D-resistant rickets, and patients with Paget's disease of bone before and after they had been made hyperphosphataemic by treatment with the drug ethylidene-1-hydroxy-1,1-bisphosphonate. Changes in intramuscular P1 were estimated from the ratio of the areas of the Pi to adenosine 5'-triphosphate peaks. Even though the plasma Pi concentration in these patients spanned a fourfold range (0.5-2.0 mmol/l) the corresponding intramuscular Pi concentration increased by only 70%. A similar effect was observed in erythrocytes, from patients with these disorders, which were incubated in autologous plasma at 37 degrees C, under an atmosphere of O2 + CO2 (95:5, v/v). However, chloride ions, which are transported passively across the cell membrane, showed no change in distribution between cells and plasma, indicating that there was no general effect on passive anion distribution. When erythrocytes from normal subjects were incubated in autologous plasma (1.0 mmol of Pi/l) and in plasma supplemented with Pi (2.3 mmol of Pi/l), the Pi concentration in the cells, at steady state, increased only from 0.57 to 0.78 mmol/l cells, suggesting that the effect was not an artifact of disease or drug therapy. It is concluded that, in human skeletal myocytes and erythrocytes, the percentage change in the concentration of cytoplasmic Pi is lower than that in plasma. This implies that these cells can buffer or regulate cytoplasmic Pi when the extracellular concentration is disturbed.